FOXC1-mediated LINC00301 facilitates tumor progression and triggers an immune-suppressing microenvironment in non-small cell lung cancer by regulating the HIF1α pathway
FOXC1-mediated LINC00301 facilitates tumor progression and triggers an immune-suppressing microenvironment in non-small cell lung cancer by regulating the HIF1α pathway
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FOXC1 介导的 LINC00301 通过调节 HIF1 α 通路促进非小细胞肺癌的肿瘤进展并触发免疫抑制微环境
DOI：
10.1186/s13073-020-00773-y
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发表时间：
2020-09-02
期刊：
影响因子：
12.3
通讯作者：
Li, De-Jia
中科院分区：
文献类型：
--
作者：
Sun, Cheng-Cao;Zhu, Wei;Li, De-Jia
背景：长链非编码RNA（lncRNA）在各种癌症的发生和转移中广泛复杂。然而，lncRNA在非小细胞肺癌（non-small cell lung cancer，NSCLC）中的具体分子机制仍不清楚。方法：采用qPCR方法检测LINC 00301在NSCLC临床标本或细胞系中的表达。采用荧光原位杂交（FISH）技术检测LINC 00301在NSCLC细胞中的定位。用染色质免疫沉淀法（ChIP）验证FOXC 1与LINC 00301启动子的结合活性。进行RNA免疫沉淀（RIP）以探索LINC 00301与EZH 2之间的结合活性。进行RNA下拉，然后进行点印迹、蛋白质结构域作图和RNA电泳迁移率变动分析（EMSA）以鉴定LINC 00301和EZH 2之间的详细结合区域。结果：LINC 00301在NSCLC组织中高表达，差异表达的lncRNA与预后密切相关。体内外实验表明，LINC 00301促进NSCLC细胞增殖，解除NSCLC细胞周期阻滞，促进细胞迁移和侵袭，抑制NSCLC细胞凋亡。此外，LINC 00301通过靶向TGF-β增加LA-4/SLN-205衍生肿瘤中的调节性T细胞（Treg），同时减少CD 8（+）T细胞群。转录因子FOXC 1介导LINC 00301在NSCLC中的表达。生物信息学预测和体外实验表明LINC 00301（83-123核苷酸[nt]）可直接与EZH 2（612-727氨基酸[aa]）的增强子结合，启动EAF 2启动子上的H3 K27 me 3。EAF 2直接结合并稳定von Hippel-Lindau蛋白（pVHL），因此下调的EAF 2通过调节NSCLC细胞中的pVHL来增强低氧诱导因子1 α（HIF 1 α）的表达。此外，我们还发现LINC 00301可以作为一种竞争性内源性RNA（ceRNA）对抗miR-1276，从而加速HIF 1 α在NSCLC细胞胞质中的表达。总之，我们目前的研究揭示了LINC 00301在临床标本以及细胞和动物实验中的致癌作用，阐明了FOXC 1/LINC 00301/EZH 2/EAF 2/pVHL/HIF 1 α和FOXC 1/LINC 00301/miR-1276/HIF 1 α通路的潜在作用和机制，为NSCLC的治疗提供了新的见解和潜在的靶点。
Background: Long non-coding RNAs (lncRNAs) are extensively intricate in the tumorigenesis and metastasis of various cancer types. Nevertheless, the detailed molecular mechanisms of lncRNA in non-small cell lung cancer (NSCLC) still remain mainly undetermined.Methods: qPCR was performed to verify LINC00301 expression in NSCLC clinical specimens or cell lines. Fluorescence in situ hybridization (FISH) was conducted to identify the localization of LINC00301 in NSCLC cells. Chromatin immunoprecipitation (ChIP) was subjected to validate the binding activity between FOXC1 andLINC00301promoters. RNA immunoprecipitation (RIP) was performed to explore the binding activity between LINC00301 and EZH2. RNA pull-down followed by dot-blot, protein domain mapping, and RNA electrophoresis mobility shift assay (EMSA) were conducted to identify the detailed binding regions between LINC00301 and EZH2. Alpha assay was conducted to quantitatively assess the interaction between LINC00301 and EZH2.Results: LINC00301 is highly expressed in NSCLC and closely corelated to its prognosis by analyzing the relationship between differentially expressed lncRNAs and prognosis in NSCLC samples. in vitro and in vivo experiments revealed that LINC00301 facilitates cell proliferation, releases NSCLC cell cycle arrest, promotes cell migration and invasion, and suppresses cell apoptosis in NSCLC. In addition, LINC00301 increases regulatory T cell (Treg) while decreases CD8(+)T cell population in LA-4/SLN-205-derived tumors through targeting TGF-beta. The transcription factor FOXC1 mediates LINC00301 expression in NSCLC. Bioinformatics prediction and in vitro experiments indicated that LINC00301 (83-123 nucleotide [nt]) can directly bind to the enhancer of zeste homolog 2 (EZH2) (612-727 amino acid [aa]) to promote H3K27me3 at theELL protein-associated factor 2(EAF2) promoter. EAF2 directly binds and stabilizes von Hippel-Lindau protein (pVHL), so downregulated EAF2 augments hypoxia-inducible factor 1 alpha (HIF1 alpha) expression by regulating pVHL in NSCLC cells. Moreover, we also found that LINC00301 could function as a competing endogenous RNA (ceRNA) against miR-1276 to expedite HIF1 alpha expression in the cytoplasm of NSCLC cells.Conclusions: In summary, our present research revealed the oncogenic roles of LINC00301 in clinical specimens as well as cellular and animal experiments, illustrating the potential roles and mechanisms of the FOXC1/LINC00301/EZH2/EAF2/pVHL/HIF1 alpha and FOXC1/LINC00301/miR-1276/HIF1 alpha pathways, which provides novel insights and potential theraputic targets to NSCLC.